Bottom Line:
We found that the IEP was localized in the nucleus and nucleolus of the cells.Remarkably, it also accumulated at the periphery of the nuclear matrix.We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

ABSTRACTGroup II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

f2: Subcellular distribution of FLAG-tagged IEP in HeLa cells.Immunofluorescence and bright-field (BF) microscopy of HeLa HA cells transfected with the constructs shown on the right. The domains of the IEP are indicated as follows: in pink, the reverse transcriptase; in green, the maturase and, in yellow, the C-terminal domain. Immunolocalization of FLAG-IEP, in green (Flag column). The nucleolus, detected with anti-fibrillarin antibody, is shown in red. A merged image of FLAG-IEP and the nucleolus is shown (merge column). DAPI (blue) was used to stain nuclear DNA. The numbers in the panels indicate the percentage of transfected cells displaying the corresponding fluorescence pattern. White arrowheads indicate signal surrounding the nuclear matrix.

Mentions:
For confirmation of the subcellular distribution of the RmInt1 IEP, and to check that there were no limitations associated with the use of YFP-fusion proteins, we then analyzed the subcellular distribution of FLAG-tagged IEP in cultured HeLa cells. We added a FLAG epitope to the N-terminus of the RmInt1 IEP and expressed the tagged-protein in the mammalian expression plasmid pCEP4, with expression under the control of the CMV (Pol II) promoter. As controls, we also generated N-terminal FLAG versions of mutated forms of the RmInt1 IEP. Following transfection, we found that the FLAG-tagged IEP was located in the nucleus, mostly in the nucleolus, but also at the periphery of the nuclear matrix, in 90% of the transfected cells (Fig. 2a). In the remaining 10% of the cells, the staining formed foci throughout the cell. Similar results were obtained with pCEP4flagIEPΔORF, in which the IEP was co-expressed with the RmInt1 ribozyme (Fig. 2b). The nucleolar localization of the FLAG-tagged IEP was further confirmed by colocalization experiments with an anti-fibrillarin antibody as a nucleolar marker. Thus, YFP seems to be primarily responsible for the exclusion of the IEP from the nucleolus. Furthermore, we speculate that localization to the periphery of the nuclear matrix may result from the direct association of the IEP with the inner nuclear membrane components or the nuclear lamina. The latter is absent from plant cells, potentially accounting for the distribution observed in Arabidopsis protoplasts, in which the RmInt1 IEP was also found in the nucleolus24, but not at the periphery of the nucleus.

f2: Subcellular distribution of FLAG-tagged IEP in HeLa cells.Immunofluorescence and bright-field (BF) microscopy of HeLa HA cells transfected with the constructs shown on the right. The domains of the IEP are indicated as follows: in pink, the reverse transcriptase; in green, the maturase and, in yellow, the C-terminal domain. Immunolocalization of FLAG-IEP, in green (Flag column). The nucleolus, detected with anti-fibrillarin antibody, is shown in red. A merged image of FLAG-IEP and the nucleolus is shown (merge column). DAPI (blue) was used to stain nuclear DNA. The numbers in the panels indicate the percentage of transfected cells displaying the corresponding fluorescence pattern. White arrowheads indicate signal surrounding the nuclear matrix.

Mentions:
For confirmation of the subcellular distribution of the RmInt1 IEP, and to check that there were no limitations associated with the use of YFP-fusion proteins, we then analyzed the subcellular distribution of FLAG-tagged IEP in cultured HeLa cells. We added a FLAG epitope to the N-terminus of the RmInt1 IEP and expressed the tagged-protein in the mammalian expression plasmid pCEP4, with expression under the control of the CMV (Pol II) promoter. As controls, we also generated N-terminal FLAG versions of mutated forms of the RmInt1 IEP. Following transfection, we found that the FLAG-tagged IEP was located in the nucleus, mostly in the nucleolus, but also at the periphery of the nuclear matrix, in 90% of the transfected cells (Fig. 2a). In the remaining 10% of the cells, the staining formed foci throughout the cell. Similar results were obtained with pCEP4flagIEPΔORF, in which the IEP was co-expressed with the RmInt1 ribozyme (Fig. 2b). The nucleolar localization of the FLAG-tagged IEP was further confirmed by colocalization experiments with an anti-fibrillarin antibody as a nucleolar marker. Thus, YFP seems to be primarily responsible for the exclusion of the IEP from the nucleolus. Furthermore, we speculate that localization to the periphery of the nuclear matrix may result from the direct association of the IEP with the inner nuclear membrane components or the nuclear lamina. The latter is absent from plant cells, potentially accounting for the distribution observed in Arabidopsis protoplasts, in which the RmInt1 IEP was also found in the nucleolus24, but not at the periphery of the nucleus.

Bottom Line:
We found that the IEP was localized in the nucleus and nucleolus of the cells.Remarkably, it also accumulated at the periphery of the nuclear matrix.We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

ABSTRACTGroup II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.